The gram-negative bacterium, Actinobacillus actinomycetemcomitans, has been the subject of intensive study because it is considered to be a major pathogen of periodontal diseases. Some of the hallmarks of the pathogenic potential of this species are the ability of select strains to invade gingival tissues and to produce relatively high levels of leukotoxin. A number of less-characterized virulence potentials include a variety of cytostatic and cytotoxic factors. The focus of this application is a novel toxin, known as cytolethal distending toxin (CDT), which the Principal Investigator has recently discovered in A. actinomycetemcomitans. In preliminary experiments, the cdt locus was cloned in a non-enteropathogenic strain of Escherichia coli and the complete nucleotide sequence obtained. Extracts of the recombinant cells caused morphological changes in Chinese hamster ovary (CHO) cells, human epithelial cell-like lines and periodontal ligament fibroblasts characteristic of CDT. The major objective of this study is to initiate an investigation towards the complete genetic and biochemical characterization of the CDT of A. actinomycetemcomitans. Three specific aims are proposed. Aim 1 is to identify and characterize the genes and gene products of the putative cdt locus. Aim 2 is to determine the kinetics of cytostatic and cytolytic effects of CDT on cultured cells, and Aim 3 is to examine the genetic organization of the cdt locus. Frame shift mutants will be constructed and employed to examine the contribution of each of the cdt genes to distension and killing activities. The kinetics of cell distension and cytotoxicity will be characterized using CHO cells as a model system. Northern blotting and primer extension will be used to determine if the three genes that comprise the cdt locus are organized into a polycistronic operon. The CDT represents a previously unknown potential virulence factor for A. actinomycetemcomitans. The expression of this novel toxin may have important implications in the development of localized juvenile periodontitis (LJP), as well as in other types of bacterial disease, because it extends the repertoire of virulence potentials of the bacterium. Significant advances have been made in the identification and description of this toxin locus and it is now important to extend our understanding of its expression and biological activities. The availability of the cloned and sequenced cdt genes provides an advantage in the development of tools for the study of the role of this toxin in the pathogenicity of oral disease.